Antibiotics XK-62-3 and XK-62-4 and process for production thereof

ABSTRACT

New antibacterial compounds, XK-62-3 and XK-62-4, are produced by fermentation of microorganisms belonging to the genus Micromonospora. The compounds are accumulated in the culture liquor and are isolated therefrom.

BACKGROUND OF THE INVENTION

The present invention relates to new compositions of matter havingantibacterial activity and more specifically to new antibioticsdesignated XK-62-3 and XK-62-4. The invention also pertains to theproduction of XK-62-3 and/or XK-62-4 by culturing a microorganismbelonging to the genus Micromonospora, which is capable of producing oneor both of the active substances in a nutrient medium, untilantibacterial activity is detected in the culture liquor and thenisolating at least one of the active substances therefrom.

Antibiotics which exhibit activity against a broad spectrum of bacteriaare always in demand. To this end, it is known that when certain strainsof Micromonospora are cultured in a nutrient medium, several antibioticsubstances are liberated in the culture liquor. One such substance isXK-62-2 which is isolated from the culture liquor of Micromonosporasagamiensis MK-65, ATCC 21826 (FERM-P No. 1530), Micromonosporasagmiensis var. nonreducans MK-62, ATCC 21803 (FERM-P No. 1477) and thelike. XK-62-2 has the following structural formula: ##STR1##

The chemical, physical and biological properties of XK-62-2 and aprocess for the production thereof are described in U.S. Pat. No.4,045,298 issued Aug. 30, 1977.

It has now been found that a new mutant strain of Micromonosporasagamiensis, when cultured, liberates, in addition to XK-62-2, twofurther active substances. A study of the chemical, physical andbiological properties of these active substances indicates that thecompositions of matter are new antibiotics which are designated XK-62-3and XK-62-4.

SUMMARY OF THE INVENTION

In accordance with the present invention, novel antibiotics, XK-62-3 andXK-62-4, are produced by fermentation of a microorganism belonging tothe genus Micromonospora, which is capable of producing one or both ofsaid antibiotics in a nutrient medium until substantial antibacterialactivity is detected in the culture liquor. At the completion ofculturing, the active fractions containing XK-62-3 or XK-62-4 areisolated from the culture liquor such as by ion exchange resintreatment.

The novel compositions of matter of the present invention arerepresented by the following general formula: ##STR2## wherein R¹ is Hwhen R² is CH₃ and R¹ is CH₃ when R² is H.

XK-62-3 and XK-62-4 exhibit broad antibacterial activity, and are,therefore, useful inter alia to clean and sterilize laboratory glasswareand surgical instruments, and may also be used in combination withsoaps, detergents and wash solutions for sanitation purposes. Further,XK-62-3 and KX-62-4 are expected to be useful as therapeutic compoundsin connection with infections induced by various bacteria.

Included in the composition of matter aspect of the invention are thepharmaceutically acceptable non-toxic acid addition salts of XK-62-3 andXK-62-4 including the mineral acid addition salts such as hydrochloride,hydrobromide, hydroiodide, sulfate, sulfamate, phosphate, carbonate andnitrate and the organic acid addition salts such as maleate, acetate,citrate, oxalate, succinate, benzoate, tartrate, fumarate, malate,mandelate, ascorbate and the like.

DETAILED DESCRIPTION OF THE INVENTION

The physicochemical properties of the free base of the antibioticXK-62-3 of the present invention are as follows.

(1) Basic white powder

(2) Elementary analytical values found: C=49.77%, H=9.02%, N=14.26%

(3) Melting point: 95°-103° C.

(4) Ultraviolet absorption spectrum of an aqueous solution of XK-62-3shows no characteristic absorption maximum between 220 nm and 360 nm butonly shows terminal absorption.

(5) Specific rotation: [α]_(D) ²⁵ =+182° (c=0.5, H₂ O)

(6) Infrared absorption spectrum, measured in KBr tablet, shows maximumabsorption at the following wavenumbers (cm⁻¹): 3330, 2920, 1590, 1450,1360, 1040, 1020

    ______________________________________                                        (7)      Color reactions                                                      ______________________________________                                               Ninhydrine test:  positive                                                    Potassium permanganate test:                                                                    positive                                                    Elson-Morgan's test                                                                             negative                                                    Biuret test:      negative                                             ______________________________________                                    

(8) PMR spectrum, measured in a deuterium oxide solution (pD=10.2),using a JEOL JNM-PS-100 spectrometer:

δ (ppm) 1.21(3H, s), 0.8-1.95(6H, m), 1.95-3.05 (6H, m), 2.34(3H, s),2.54(3H, s), 3.05-4.20(7H, m), 5.06(1H, d, J=4), 5.20(1H, d, J=4)

(9) CMR spectrum, measured in a deuterium oxide solution (pD=11.0),using a JEOL-PFT-100A spectrometer:

δ (ppm) 102.4, 101.1, 87.4, 85.8, 75.4, 73.3, 71.7, 70.2, 68.5, 64.2,57.9, 51.4, 50.6, 45.8, 37.8, 32.8, 32.6, 28.2, 26.8, 22.5

(10) Mass spectrum of XK-62-3 reveals the following M+1 ion and fragmentions. The formulae in parentheses are the composition formulae obtainedby high resolution mass spectrometry.

m/e 464 M+1 (C₂₀ H₄₂ N₅ O₇), 446(C₂₀ H₃₈ N₄ O₇), 388(C₁₇ H₃₄ N₅ O₅),364(C₁₅ H₃₀ N₃ O₇), 346(C₁₅ H₂₈ N₃ O₆), 336(C₁₄ H₃₀ N₃ O₆), 333(C₁₄ H₂₉N₄ O₅), 318(C₁₄ H₂₈ N₃ O₅), 289(C₁₃ H₂₅ N₂ O₅), 272(C₁₃ H₂₆ N₃ O₃), 205(C₈ H₁₇ N₂ O₄), 177(C₇ H₁₇ N₂ O₃), 160(C₇ H₁₄ NO₃), 129(C₆ H₁₃ NO₂).

From the foregoing, it is determined that the molecular weight of thesubstance is 463 and that the molecular formula is C₂₀ H₄₁ N₅ O₇. Theelementary analytical values of the substance (hydrated with 1 mole ofH₂ O) as calculated from the molecular formula are C=49.88%, H=9.00% andN=14.54%.

(11) From the foregoing physicochemical properties, the antibioticXK-62-3 is determined to have the following structural formula: ##STR3##

The free base of XK-62-3 is very soluble in water, soluble in methanoland slightly soluble in ethanol and acetone but insoluble in organicsolvents such as chloroform, benzene, ethyl acetate, butyl acetate,ether, butanol, petroleum ether, n-hexane, and the like.

The physicochemical properties of the free base of the antibioticXK-62-4 of the present invention are as follows:

(1) Basic white powder

(2) Elementary analytical values found: C=51.61%, H=9.28%, N=14.18%

(3) Melting point: 101°-112° C.

(4) Ultraviolet absorption spectrum of an aqueous solution of XK-62-4shows no characteristic absorption maximum between 220 nm and 360 nm butonly shows terminal absorption.

(5) Specific rotation: [α]_(D) ²⁵ =+143° (c=0.5, H₂ O)

(6) Infrared absorption spectrum, measured in KBr tablet, showsabsorption maximum at the following wavenumbers (cm⁻¹): 3350, 2920,1570, 1460, 1340, 1050, 1020

    ______________________________________                                        (7)      Color reactions                                                      ______________________________________                                               Ninhydrine test:  positive                                                    Potassium permanganate test:                                                                    positive                                                    Elson-Morgan's test                                                                             negative                                                    Biuret test:      negative                                             ______________________________________                                    

(8) PMR spectrum, measured in a deuterium oxide solution (pD=10.3),using a JEOL JNM-PS-100 spectrometer:

δ (ppm) 1.21(3H, s), 0.8-2.10(6H, m), 2.24(6H, s), 2.35-2.68(3H, m),2.34(3H, s), 2.68-3.05(3H, m) 3.05-4.20(7H, m), 5.07(1H, d, J=4),5.17(1H, d, J=4)

(9) CMR spectrum, measured in a deuterium oxide solution (pD=10.5),using a JEOL-PFT-100A spectrometer:

δ (ppm) 101.2, 101.2, 87.6, 87.4, 75.1, 73.2, 70.1, 68.5, 67.6, 64.2,63.8, 51.6, 50.6, 50.4, 45.7, 45.7, 37.7, 36.4, 29.3, 26.5, 22.4

(10) Mass spectrum of XK-62-4 reveals the following molecular ion andfragment ions. The formulae in parentheses are the composition formulaeobtained by high resolution mass spectrometry.

m/e 477 M (C₂₁ H₄₃ N₅ O₇), 460(C₂₁ H₄₀ N₄ O₇), 360(C₁₆ H₃₂ N₄ O₅),350(C₁₄ H₂₈ N₃ O₇), 347(C₁₅ H₃₁ N₄ O₅), 322(C₁₃ H₂₈ N₃ O₆), 304(C₁₃ H₂₆N₃ O₅), 286(C₁₄ H₂₈ N₃ O₃), 160(C₇ H₁₄ NO₃), 157(C₈ H₁₇ N₂ O).

From the foregoing, it is determined that the molecular weight of thesubstance is 477 and that the molecular formula is C₂₁ H₄₃ N₅ O₇. Theelementary analytical values of the substance (hydrated with 1/2 mole ofH₂ O) as calculated from the molecular formula are C=51.83%, H=9.11% andN=14.39%.

(11) From the foregoing physicochemical properties, the antibioticXK-62-4 is determined to have the following structural formula: ##STR4##

The free base of XK-62-4 is very soluble in water, soluble in methanoland slightly soluble in ethanol and acetone but insoluble in suchorganic solvents as chloroform, benzene, ethyl acetate, butyl acetate,ether, butanol, petroleum ether, n-hexane, and the like.

The Rf values of XK-62-3 and XK-62-4 in paper chromatography usingvarious developers are set forth in the following Tables 1 to 3. Forcomparison, the Rf values of antibiotics which are considered to besimilar to XK -62-3 and XK-62-4 are also given.

                  TABLE 1                                                         ______________________________________                                        Rf values in ascending paper chromatography (at 28° C.)                                                    Period of                                                                     devel-                                                                        opment                                    Developer         XK-62-3  XK-62-4  (hours)                                   ______________________________________                                        20% ammonium chloride                                                                           0.96     0.96     3                                         water-saturated n-butanol                                                                       0.00     0.00     15                                        n-butanol . acetic acid . water                                                                 0.05     0.05     15                                        (3:1:1)                                                                       (by volume)                                                                   water-saturated ethyl acetate                                                                   0.00     0.00     4                                         water-saturated n-butanol                                                                       0.10     0.10     15                                        containing 2% (W/V) p-toluene                                                 sulfonic acid and 2% (W/V)                                                    piperidine                                                                    ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Rf values in silica gel thin layer                                            chromatography (at room temperature;                                          after three hours of development)                                             Developer*   Antibiotic      Rf value                                         ______________________________________                                        I            XK-62-3         0.67                                             "            XK-62-4         0.71                                             "            Fortimicin A    0.74                                             "            Fortimicin B    0.80                                             "            Fortimicin C    0.75                                             "            Fortimicin D    0.74                                             "            Fortimicin KE   0.78                                             "            Gentamicin complex                                                                            0.71                                             "            Gentamicin C.sub.1a                                                                           0.71                                             "            Sisomicin       0.71                                             II           XK-62-3         0.06                                             "            XK-62-4         0.10                                             "            Fortimicin A    0.37                                             "            Fortimicin B    0.62                                             "            Fortimicin C    0.40                                             "            Fortimicin D    0.37                                             "            Fortimicin KE   0.58                                             "            Gentamicin complex                                                                            0.06-0.16                                        "            Gentamicin C.sub.1a                                                                           0.16                                             "            Sisomicin       0.18                                             ______________________________________                                         *Developer I: The upper layer of chloroform, methanol and 17% (w/w)           aqueous ammonia (2:1:1, by volume).                                           Developer II: 10% ammonium acetate and methanol (1:1, by volume).        

                  TABLE 3                                                         ______________________________________                                        Rf values in ascending paper chromatography                                   using the lower layer of chloroform, methanol                                 and 17% (w/w) aqueous ammonia (2:1:1 by volume)                               as the developer, and Whatman No. 1 (W. & R.                                  Balston Ltd., United Kingdom) as the paper (at                                room temperature; after 12 hours of development).                             Antibiotics            Rf value                                               ______________________________________                                        Streptomycin A         0.02                                                   Streptomycin B         0.00                                                   Bluensomycin           0.01                                                   Ribostamycin           0.00                                                   Lividomycin A          0.00                                                   Lividomycin B          0.03                                                   Hygromycin B           0.02                                                   Lividomycin D          0.02                                                   Spectinomycin          0.45                                                   Kasugamycin            0.01                                                   Butirosine A           0.00                                                   Butirosine B           0.01                                                   Gentamicin A           0.00                                                   Gentamicin B           0.00                                                   Gentamicin C.sub.1a    0.18                                                   Gentamicin C.sub.1     0.59                                                   Gentamicin C.sub.2     0.38                                                   Sisomicin              0.18                                                   Neomycin A             0.00                                                   Neomycin B             0.03                                                   Antibiotic No. 460     0.01                                                   Neomycin C             0.00                                                   Kanamycin A            0.02                                                   Kanamycin B            0.01                                                   Kanamycin C            0.02                                                   Paromomycin            0.00                                                   Nebramycin complex     0.01                                                   Tobramycin             0.02                                                   Apramycin              0.02                                                   Nebramycin Factor 4    0.01                                                   Nebramycin Factor 5    0.00                                                   Myomycin               0.00                                                   Seldomycin Factor 1    0.00                                                   Seldomycin Factor 2    0.01                                                   Seldomycin Factor 3    0.00                                                   Seldomycin Factor 5    0.09                                                   XK-62-2                0.49                                                   Fortimicin B           0.65                                                   Fortimicin A           0.37                                                   Fortimicin C           0.18                                                   Fortimicin D           0.18                                                   Fortimicin KE          0.59                                                   XK-62-3                0.40                                                   XK-62-4                0.70                                                   ______________________________________                                    

Table 4 illustrates the antibacterial spectra of XK-62-3 and XK-62-4against various microorganisms.

                  TABLE 4                                                         ______________________________________                                        (Minimum Inhibitory Concentration, γ/ml                                 measured by agar dilution method at pH 8.0)                                                          XK-62-3  XK-62-4                                       Microorganism          γ/ml                                                                             γ/ml                                    ______________________________________                                        Bacillus subtilis No. 10707                                                                          <0.0011  <0.0011                                       Staphylococcus aureus ATCC 6538P                                                                     0.0021   0.0021                                        Klebsiella pneumoniae ATCC 10031                                                                     0.0082   0.0082                                        Klebsiella pneumoniae KY 4261                                                                        0.066    0.066                                         Escherichia coli ATCC 26                                                                             0.066    0.033                                         Escherichia coli KY 8310                                                                             0.066    0.066                                         Escherichia coli KY 8327                                                                             0.066    --                                            [resistant to kanamycin, gentamicin                                           and tobramycin, ANT(2")]*.sup.1                                               Escherichia coli KY 8332                                                                             --       0.033                                         [resistant to kanamycin, AAC(6')-I]*.sup.2                                    Escherichia coli KY 8348                                                                             0.033    --                                            [resistant to gentamicin, AAC(3)-I]*.sup.3                                    Escherichia coli KY 8356                                                                             0.26     --                                            Pseudomonas aeruginosa BMH No. 1                                                                     0.26     0.26                                          Pseudomonas aeruginosa KY 8563                                                                       0.25     >10                                           [resistant to gentamicin, AAC(3)-II*.sup.4                                    Pseudomonas aeruginosa KY 8511                                                                       0.26     --                                            [resistant to gentamicin, AAC(3)-I]*.sup.3                                    Pseudomonas aeruginosa Z 444                                                                         >10      0.26                                          [resistant to gentamicin, AAC(6')-III] *.sup.5                                Pseudomonas aeruginosa Z 445                                                                         --       0.26                                          [resistant to gentamicin, AAC(6')-III]*.sup.5                                 Proteus vulgaris ATCC 6897                                                                           0.26     0.13                                          Shigella sonnei ATCC 9290                                                                            0.13     0.066                                         Salmonella typhosa ATCC 9992                                                                         0.033    0.011                                         Serratia marcescens KY 4248                                                                          1.52     0.26                                          Providenicia sp. KY 8464                                                                             1.04     --                                            ______________________________________                                         *.sup.1 ANT(2"): resistant strain by 2adenylation                             *.sup.2 AAC(6')I: resistant strain by 6N-acetylation (Type I)                 *.sup.3 ACC(3)I: resistant strain by 3N-acetylation (Type I)                  *.sup.4 AAC(3)II: resistant strain by 3N-acetylation (Type II)                *.sup.5 AAC(6')III: resistant strain by 6N-acetylation (Type III)        

As is apparent from the foregoing, the antibiotics XK-62-3 and XK-62-4have a very strong antibacterial activity against a broad range ofGram-positive and Gram-negative bacteria. The compounds are particularlyeffective upon microorganisms of the genera Proteus and Pseudomonas uponwhich only a few antibiotics have been known to be effective.Furthermore, XK-62-3 has a strong antibacterial activity against certainstrains of Escherichia coli and Pseudomonas aeruginosa which areresistant to various known antibiotics and particularly against certainresistant strains which have mechanisms of inactivation such as3-N-acetylation, 2"-adenylation, etc. Therefore, XK-62-3 is more usefulthan gentamicin, sagamicin (antibiotic XK-62-2), kanamycin and the likewhich are ineffective on those resistant strains which have theabove-mentioned mechanisms of inactivation.

On the other hand, XK-62-4 also has a strong antibacterial activityagainst certain strains of Escherichia coli and Pseudomonas aeruginosawhich are resistant to various known antibiotics and particularlyagainst those resistant strains which have mechanisms of inactivationsuch as 6'-N-acetylation, etc. Therefore, XK-62-4 is more useful thansisomicin, dibekacin (3,4-dideoxy kanamycin B), kanamycin A and the likewhich are ineffective on those resistant strains which have theabovementioned mechanisms of inactivation.

The acute toxicities, LD₅₀ (mg/kg), of the compounds XK-62-3 and XK-62-4determined using dd-mice weighing 20±1 g are 106 and 25.9 mg/kg,respectively.

A comparison of XK-62-3 with known antibiotics further illustrates theirnovelty. As water-soluble, basic antibiotics produced by microorganismsof the genus Micromonospora and having a broad antibacterial spectra,there are the gentamicin complex (M. J. Weinstein et al: AntimicrobialAgents and Chemotherapy, 1963, 1; D. J. Cooper et al: J. Infect. Dis.119, 342, 1969; and J. A. Waitz et al: Antimicrobial Agents andChemotherapy 2, 464, 1972), antibiotic No. 460 (Japanese PatentPublication No. 46-16153), sisomicin (M. J. Weinstein et al: J.Antibiotics, 23, 551, 555, 559, 1970), XK-62-2 (U.S. Pat. No.4,045,298), fortimicin B (U.S. Pat. No. 3,931,400), fortimicin A (U.S.Pat. No. 3,976,768), fortimicin C (U.S. Pat. No. 4,048,015), andfortimicin D and KE (U.S. patent application Ser. No. 845,970).

However, from the Rf values in paper chromatography shown in Table 3, itis evident that XK-62-3 and XK-62-4 are different from such knownantibiotics.

In addition, as water-soluble, basic antibiotics produced byActinomycetes other than those of the genus Micromonospora and having abroad antibacterial spectra, streptomycin A and B, ribostamycin,lividomycin A, B and D, neomycin A, B and C, kanamycin A, B and C,nebramycin complex, nebramycin factors 4 and 5 and paromycin are known.XK-62-3 and XK-62-4 have been found to be greatly different from any ofthese antibiotics in physicochemical properties. Moreover, as isapparent from Table 3, XK-62-3 and XK-62-4 are quite different fromthese antibiotics in Rf values.

From the foregoing, XK-62-3 and XK-62-4 are considered to be newantibacterial compounds.

XK-62-3 and XK-62-4 are produced by fermentation of a novelmicroorganism belonging to the genus Micromonospora, namelyMicromonospora sagamiensis var nonreducans KY 11504 (NRRL-11101)(FERM-P, No. 3962). The strain has been deposited as Micromonosporasagamiensis KY 11504 with the United States Department of Agriculture,Peoria, Illinois, and with the Fermentation Research Institute Agency ofIndustrial Science and Technology, Tokyo, Japan and has been accordedthe accession numbers noted above.

The novel microorganism of the present invention was obtained by doublemutation of Micromonospora sagamiensis var nonreducans MK-62, ATCC21803, a known XK-62-2 producing organism. The microbiologicalproperties of the parent strain are set forth in detail in U.S. Pat. No.4,045,298 which description is expressly incorporated herein byreference.

Briefly, the mutation treatment by which the strain useful for thepresent invention was obtained is as follows. Cells of Micromonosporasagamiensis var nonreducans ATCC 21803 were incubated in a 1 mg/mlsolution of N-methyl-N'-nitro-N-nitrosoguanidine for 18 hours. Thesuspension was then smeared on an agar plate and, after incubation, theresulting colonies were picked up. Cultures of the respective colonieswere then tested for productivity of antibacterial substances and onestrain showing an increased ability to produce XK-62-2 was isolated.This strain is also identified in the aforementioned U.S. Pat. No.4,045,298 as Micromonospora sagamiensis var nonreducans MK-62-NG-164(ATCC 21949) which description is also incorporated herein by reference.The latter strain was then subjected to gamma-ray irradiation. Afterirradiation, the culture was smeared on an agar plate and incubated. Theresulting colonies were picked up and tested for ability to produceactive substances; and the strain of the instant invention was isolatedas a biologically pure culture. As such, the strain has the ability toproduce XK-62-3 and XK-62-4 in recoverable quantities. Morphologically,the identifying characteristics of the strain of the present inventionare identical to the parent strains except that visually, the mutantappears to bear slightly less spores.

Those skilled in the art will appreciate that although the instantstrain was obtained by a double mutation treatment, other microorganismsuseful in carrying out the present invention may be obtained by a singlemutation treatment by artificial means such as ultraviolet irradiation,X-ray irradiation and use of various mutation including chemicals inknown manner to enhance the production of metabolic products andparticularly the composition of matter of the invention.

Generally, conventional methods for culturing Actinomycetes may beemployed in the process of the present invention. Thus, various nutrientsources may be used for the culture medium. Appropriate carbon sourcesinclude glucose, starch, mannose, dextrin, fructose, sucrose, molasses,etc. either alone or in combination. Hydrocarbons, alcohols, organicacids, etc. may also be used depending upon the assimilability possessedby the microorganisms to be used. As inorganic and organic nitrogensources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate,sodium nitrate, may be used either alone or in combination or naturalnitrogen sources such as peptone, meat extract, yeast extract, dryyeast, corn steep liquor, soybean powder, casamino acid, solublevegetable protein, etc. are appropriate. If necessary, inorganic saltssuch as sodium chloride, potassium chloride, calcium carbonate,phosphates, etc. may be added to the medium. Moreover, organic andinorganic materials which promote the growth of the particular strainand the production of XK-62-3 and/or XK-62-4 may be added.

A liquid culturing method, particularly a submerged stirring culturingmethod is most suitable. Culturing temperature is desirably 25°-40° C.,and it is preferred to carry out culturing at around neutral pH.Usually, after 2 to 15 days of liquid culturing, XK-62-3 and/or XK-62-4are formed and accumulated in the culture liquor. When the yield of theantibiotics in the culture liquor reaches a maximum, culturing isdiscontinued and the desired product is isolated and purified from theculture liquor after the microbial cells have been removed such as byfiltration.

Isolation and purification of XK-62-3 and XK-62-4 are carried out bymethods usually used for the isolation and purification of microbialmetabolic products from a culture liquor.

Since the antibiotics are basic substances and are readily soluble inwater but poorly soluble in ordinary organic solvents, the compounds canbe purified by the methods usually used for the purification ofso-called water-soluble basic antibiotics. More specifically, XK-62-3and XK-62-4 can be purified by a combination of adsorption anddesorption from cation exchange resin, cellulose column chromatography,adsorption and desorption using a column of Sephadex LH-20, silica gelcolumn chromatography, and the like. As an example, a suitable method ofpurification of XK-62-3 and XK-62-4 from the culture liquor (usuallycontaining a mixture of XK-62-2, XK-62-3, XK-62-4 and by-products havingantibacterial activity) when a strain capable of producing theantibiotics XK-62-3 and XK-62-4 is used is as follows. The cell-freeculture filtrate is adjusted to a weakly alkaline pH and is then passedthrough a cation exchange resin such as Amberlite IRC-50 (ammonium form)(product of Rohm & Haas Co., Ltd., U.S.A.). After the resin is washedwith water, elution is carried out with 1 N aqueous ammonia. The activefractions are combined and concentrated under reduced pressure.

The concentrate is then treated with an anion exchange resin, Dowex 1×2(OH⁻ form) (product of Dow Chemical Co., U.S.A.).

The active fractions obtained by the elution are combined andconcentrated under reduced pressure. The concentrate is adjusted toabout pH 10.5 and 4 volumes of acetone is added thereto. The resultantprecipitate is removed by filtration and the filtrate is concentratedand adjusted to pH 4.5 with sulfuric acid. To this, 5-10 volumes ofmethanol is added. The precipitate is recovered by filtration and driedin vacuo to obtain a white crude powder of XK-62 series complex.

The crude powder is dissolved in water and passed through a columnpacked with a cation exchange resin such as Amberlite CG-50, type I (NH₄⁺ form) (product of Rohm & Haas Co., Ltd.) to absorb the activeprinciples thereon. After washing with water, elution is carried outwith diluted aqueous ammonia.

After several trace components are eluted, XK-62-4 is eluted. Thenelution is continued and XK-62-3 and XK-62-2 are eluted. The fractionscontaining XK-62-3 or XK-62-4 are combined and concentrated to drynessunder reduced pressure to obtain a white crude powder of XK-62-3 orXK-62-4.

Then, each of the crude powder is subject to silica gel columnchromatography using silicic acid AR (product of Mallinckrodt ChemicalWorks Co.) as silica gel. As the developer, lower layers of a mixedsolvent comprising chloroform, methanol, acetone and concentratedaqueous ammonia (2:2:2:1 by volume) for purifying XK-62-3 and a mixedsolvent comprising chloroform, methanol, concentrated aqueous ammonia(2:1:1 by volume) for purifying XK-62-4 are used. The crude powder issuspended in the solvent and introduced into the column. Elution iscarried out with the same solvent.

First, several trace components are eluted and then XK-62-3 or XK-62-4is eluted. The fractions containing XK-62-3 or XK-62-4 are combined andconcentrated under reduced pressure. The residue is dissolved in a smallamount of water and freez-dried to obtain purified XK-62-3 or XK-62-4.

In the purification processes described above, the presence of XK-62-3or XK-62-4 in the fractions is monitored by ascending paperchromatography using Whatman filter paper No. 1. Elution is carried outwith the lower layer of solvent comprising chloroform, methanol and 17%aqueous ammonia (2:1:1 by volume) as developer at room temperature for6-15 hours. The Rf values of XK-62-3 and XK-62-4 on the paperchromatogram are 0.4 and 0.7 respectively.

The free base of XK-62-3 and XK-62-4 may be further converted to anon-toxic acid addition salt such as the mono-, di-, tri-, tetra- andpenta- salts in conventional manner such as by reacting one molecule ofthe base with one to five molecules of a pharmaceutically acceptablenon-toxic acid.

Certain specific embodiments of the present invention are illustrated bythe following representative examples.

EXAMPLE 1

A. Culturing of KY 11504 Strain

Micromonospora sagamiensis var. nonreducans KY 11504, NRRL 11101, FERM-PNo. 3962 is used as the need strain. One loopful of the seed strain isinoculated into 30 ml of the first seed medium in a 250 ml--Erlenmeyerflask. The first seed medium comprises 1 g/dl dextrin, 1 g/dl glucose,0.5 g/dl peptone, 0.5 g/dl yeast extract and 0.1 g/dl calcium carbonate(pH: 7.2 before sterilization).

Culturing is carried out with shaking at 30° C. for 5 days. Then 30 mlof the seed culture is inoculated into 300 ml of the second seed mediumin a 2 L--Erlenmeyer flask provided with baffles. The second seed mediumhas the same composition as that of the first seed medium. The secondseed culturing is carried out with shaking at 30° C. for 2 days. Then,1.5 L of the second seed culture (corresponding to the content of 5flasks) is inoculated into 15 L of the third seed medium in a 30 Lglass-jar fermenter. The composition of the third seed medium isidentical to that of the first seed medium.

Culturing in the jar fermenter is carried out with aeration (15 L/min.)and stirring (350 r.p.m.) at 30° C. for 2 days. Then, 15 L of the thirdseed culture is inoculated into 150 L of the fourth seed medium in a 300L--fermenter. The composition of the fourth seed medium is identical tothat of the first seed medium. Culturing in the fermenter is carried outwith aeration (100 L/min.) and stirring (150 r.p.m.) at 30° C. for 2days. Finally, 150 L of the fourth seed culture is inoculated into 1500L of a fermentation medium comprising 5 g/dl dextrin, 3.5 g/dl soybeanmeal and 0.7 g/dl CaCO₃ (pH 7.2 before sterilization) in a 3000L--fermenter. Culturing in the fermenter is carried out with aeration(500 L/min.) and stirring (150 r.p.m.) at 30° C. for 5 days.

B. Isolation of Crude XK-62 series complex

After the completion of fermentation, the culture liquor is adjusted topH 2.0 with 12 N sulfuric acid and is stirred for an hour. Then, about20 kg of Radiolite No. 600 (filter aid, product of Showa Kagaku KogyoCo., Ltd., Japan) is added thereto and the microbial cells are removedby filtration. The filtrate is adjusted to pH 8.0 with 6 N sodiumhydroxide and passed through a column packed with about 100 L of ionexchange resin, Amberlite IRC-50 (ammonia form). The eluate isdiscarded. Active principles are adsorbed on the resin. After washingthe resin with water, the active principles are eluted out with 1 Naqueous ammonia.

The activity of the eluate is determined against Bacillus subtilis No.10707 by a paper disk method using an agar plate. Active fractions arecombined and concentrated in vacuo to about 3 L. The concentrate isadjusted to pH 8.0 with 6 N sulfuric acid and passed through a columnpacked with 3 L of an anion exchange resin, Dowex 1×2 (OH⁻ form). Thecolumn is washed with about 20 L of water and active principles areeluted with 1 N aqueous ammonia. The active fractions are combined andconcentrated to 1/10 by volume. The pH of the concentrate is adjusted to10.5 with 6 N sodium hydroxide and 4 volumes of acetone is addedthereto. The resulting precipitate is removed by filtration and theacetone layer is concentrated to 2 L. The concentrate is adjusted to pH4.5 with 6 N sulfuric acid and then 10 L of methanol is added thereto.After cooling, a white precipitate is obtained. The precipitate isseparated by filtration and washed with methanol. After drying in vacuo,about 250 g of white powder (sulfate form) is obtained.

C. Isolation and purification of XK-62-3

In this step, 200 g of the white crude powder obtained in the precedingstep B is dissolved in 500 ml of water. The solution is adjusted to pH7.5 with 6 N sodium hydroxide and passed through a column packed with 10L of cation exchange resin, Amberlite CG-50 (NH₄ ⁺ form) (product ofRohm and Haas Co., Ltd.). The eluate is discarded. The active principlesare adsorbed on the resin. After washing the resin with water, elutionis carried out with 0.2 N aqueous ammonia. The eluate is taken in 500 mlfractions and the activity and components of each of the fractions aredetermined by the paper disc method and paper chromatography describedabove. First, several trace components are eluted and then fractionscontaining XK-62-3 are eluted. The fractions are combined andconcentrated to 50 ml. The concentrate is freeze-dried to obtain 12 g ofwhite crude powder of XK-62-3.

The crude powder is then placed on the upper part of a 35 mmφ×60 cmcolumn packed with about 500 ml of silica gel (silicic acid AR; productof Mallinckrodt Chemical Works, Co.) to form a thin, uniform layer. Thesilica gel is previously suspended in a solvent comprising chloroform,methanol, acetone and concentrated aqueous ammonia (2:2:2:1 by volume)and is packed in the column to form a tight, uniform layer. The silicagel is washed with the solvent having the same composition as describedabove.

After the crude powder is charged, the same solvent as described aboveis poured gradually into the column from the top and thereafter elutionis carried out continuously at a flow rate of about 30 ml/hour. Theeluate is taken in 20 ml fractions. The active fractions are subjectedto paper chromatography using Whatman paper No. 1 to examine thecomponents eluted. The fractions containing XK-62-3 are combined andconcentrated under reduced pressure to remove the solvent.

The residue is dissolved in a small amount of water and the solution isfreeze-dried to obtain about 1.5 g of purified preparate of the freebase of XK-62-3. The preparate exhibits an activity of about 985 unit/mg(the activity of 1 mg of pure preparate corresponds to 1000 units).

D. Isolation and purification of XK-62-4

For this step, 200 g of the crude white powder (sulfate) obtained in thesame manner as in the preceding steps A and B is dissolved in 500 ml ofwater. The solution is adjusted to pH 7.5 with 6 N sodium hydroxide andpassed through a column packed with 10 L of cation exchange resin,Amberlite CG-50 (NH₄ ⁺ form). The eluate is discarded. The activeprinciples are adsorbed on the resin. After washing the resin withwater, elution is carried out with 0.2 N aqueous ammonia and the eluateis taken in 500 ml fractions. The activity and components of each of thefractions are determined by the paper disc method and paperchromatography described above. First, several trace components areeluted and then fractions containing XK-62-4 are eluted. The fractionsare combined and concentrated to 50 ml. The concentrate is freeze-driedto obtain 15 g of white crude powder of XK-62-4.

The crude powder is placed on the upper part of a 35 mmφ×60 cm columnpacked with about 500 ml of silica gel (silicic acid AR) to form a thin,uniform layer. The silica gel is previously suspended in a solventcomprising chloroform, methanol, and concentrated aqueous ammonia (2:1:1by volume) and packed in the column to form a tight, uniform layer. Thesilica gel is washed with the solvent having the same composition asdescribed above.

After the crude powder is charged, the same solvent is poured graduallyinto the column from the top and thereafter elution is carried outcontinuously at a flow rate of about 30 ml/hour. The eluate is taken in20 ml fractions. The active fractions are subjected to paperchromatography using Whatman paper No. 1 to examine the componentseluted, and the fractions containing XK-62-4 are combined andconcentrated under reduced pressure to remove the solvent.

The residue is dissolved in a small amount of water and the solution isfreeze-dried to obtain about 2.2 g of purified preparate of the freebase of XK-62-4. The preparate exhibits an activity of about 980 unit/mg(the activity of 1 mg of pure preparate corresponds to 1000 units).

EXAMPLE 2

A. Culturing

In this example, the same strain as described in Example 1 is used andthis strain is seed cultured in first through third seed mediacomprising 2 g/dl soluble starch, 0.5 g/dl NZ-amine type A, 0.5 g/dlyeast extract and 0.1 g/dl CaCO₃. One loopful of the seed culture isinoculated into 300 ml of the seed medium in a 2 L--Erlenmeyer flask.The first seed culturing is carried out with shaking at 30° C. for 4days. Thereafter, the content of three flasks of the first seed cultureis inoculated into 15 L of fresh seed medium in a 30 L-jar fermenter.The second seed culturing is carried out with aeration and stirring at30° C. for 2 days. Then 15 L of the second seed culture is transferredto a 300 L-fermenter containing 150 L of the seed medium. The third seedculturing is carried out with aeration and stirring at 30° C. for 2days. Then, 150 L of the third seed culture is transferred to a 3000L-fermenter containing 1500 L of a fermentation medium comprising 4 g/dlsoluble starch, 1 g/dl corn steep liquor, 2 g/dl soybean meal, 0.05 g/dlK₂ HPO₄, 0.05 g/dl MgSO₄.7H₂ O, 0.03 g/dl KCl, 0.005 g/dl CoCl₂.2H₂ O,and 0.1 g/dl CaCO₃. Fermentation is carried out with aeration andstirring at 30° C. for 4 days.

B. Isolation of Crude XK-62 series complex

After the completion of fermentation, the culture liquor is adjusted topH 2.0 with 12 N sulfuric acid and stirred for 30 minutes. Then, about20 kg of Radiolite No. 600 (filter aid) is added thereto and themicrobial cells are removed by filtration. The filtrate is adjusted topH 8.0 with 6 N sodium hydroxide and passed through a column packed with100 L of a cation exchange resin, Amberlite IRC-50 (NH₄ ⁺ form). Afterwashing the resin with water, the active substances are eluted out with1 N aqueous ammonia. The eluate is obtained in fractions and theactivity of each of the fractions is determined against Bacillussubtilis No. 10707 by a paper disc method using an agar plate. Theeluate is concentrated to 2 L under reduced pressure. The concentrate isadjusted to pH 4.5 with 6 N sulfuric acid and the resulting insolublesubstance is removed by filtration. To the filtrate 20 L of methanol isadded to form a white precipitate. The precipitate is separated byfiltration and washed with methanol. After drying in vacuo, 500 g ofwhite powder, of a crude XK-62 series (sulfate form) is obtained.

C. Isolation of XK-62-3

200 g of the crude powder obtained in the above step B is isolated andpurified in the same manner as described in section C of Example 1,whereby 1.1 g of purified preparate, XK-62-3 exhibiting an activity of970 units/mg is obtained.

D. Isolation of XK-62-4

200 g of the crude powder obtained in the above step B is isolated andpurified in the same manner as described in step D of Example 1, whereby1.1 g of purified preparate having the activity of 970 units isobtained.

EXAMPLE 3

In this example, 1 g. of free base XK-62-3 is dissolved in 5 ml ofwater. Then 1.8 ml of 6 N sulfuric acid is added to the solution and 100ml of methanol is added to form a white precipitate. The whiteprecipitate is separated by filtration and washed with methanol. Theprecipitate is dried in vacuo to obtain 1.3 g of the sulfate salt ofXK-62-3. The activity of the substance is about 630 units.

EXAMPLE 4

In this example, 1 g. of free base of XK-62-4 is dissolved in 5 ml ofwater. Then 1.8 ml of 6 N sulfuric acid is added to the solution and 100ml of methanol is added to form a white precipitate. The whiteprecipitate is separated by filtration and washed with methanol. Theprecipitate is dried in vacuo to obtain 1.2 g of the sulfate salt ofXK-62-4. The activity of the substance is about 625 units.

What is claimed is:
 1. A process for producing antibiotic compoundsXK-62-3 and XK-62-4, which comprises culturing a microorganism of thegenus Micromonospora and which is capable of producing at least one ofsaid compounds in a nutrient medium until substantial antibacterialactivity is detected in the culture liquor and thereafter isolating atleast one of said compounds therefrom.
 2. A process according to claim 1wherein XK-62-3 is isolated from said culture liquor.
 3. A processaccording to claim 1 wherein XK-62-4 is isolated from said cultureliquor.
 4. A process according to claim 1 wherein said microorganism isa member of the species Micromonospora sagamiensis var. nonreducans. 5.A process according to claim 4 wherein said microorganism isMicromonospora sagamiensis var. nonreducans NRRL
 11101. 6. A processaccording to claim 1 wherein said culturing step is carried out at 25°to 40° C. for 2 to 15 days at about neutral pH.
 7. A process forproducing XK-62 series antibiotics which comprises culturingMicromonospora sagamiensis var. nonreducans NRRL 11101 in a nutrientmedium until antibacterial activity is detected in the culture liquor.8. A process according to claim 7 wherein at least one of XK-62-3 andXK-62-4 is isolated from said culture liquor.
 9. A process for producingXK-62-3 and XK-62-4 which comprises subjecting an XK-62-2 producingmicroorganism belonging to the species Micromonospora sagamiensis to atleast one mutation inducing treatment, isolating a mutant thereof whichhas the ability to produce at least one of XK-62-3 and XK-62-4;culturing said mutant in a nutrient medium until substantialantibacterial activity is detected in the culture liquor; andthereafter, isolating at least one of said XK-62-3 and XK-62-4therefrom.
 10. A biologically pure culture of the microorganismMicromonospora sagamiensis var. nonreducans, having the identifyingcharacteristics of NRRL 11101, said culture being capable of producingthe antibiotics XK-62-3 and XK-62-4 in recoverable quantities uponfermentation in a nutrient medium.